Optical imaging and detection methods offer a number of advantages over other imaging and detection methods. Imaging of tissues, organs or whole subjects typically uses light in the red and near-infrared (NIR) ranges (600-1200 nm) to maximize tissue penetration and minimize absorption from natural biological absorbers such as hemoglobin and water and autofluorescence from biological molecules. Optical imaging may provide high sensitivity, does not require exposure of test subjects or laboratory personnel to ionizing radiation, can allow for simultaneous use of multiple, distinguishable probes (which may be important in molecular imaging), and offers high temporal and spatial resolution, which is important in functional imaging, detection, diagnostic applications, microscopy, cytometry, tissue imaging, and in vitro and in vivo imaging.
In fluorescence imaging or detection, filtered light or a laser with a defined bandwidth is used as a source of excitation light. The excitation light travels through body tissue or other analytical sample such as a microscope slide, a cell, or a multi-well plate, and when the excitation light encounters a reporter molecule (for example, a contrast agent, sensitizer, fluorochrome or imaging probe), the light is absorbed. The reporter molecule then emits light, or transfers excitation signal or energy to another molecule that can emit light, that has detectably different properties from the excitation light. The resulting emitted light then can be used to construct an image or quantify the amount of reporter in the sample. Most optical imaging techniques have relied on the use of organic and inorganic fluorescent dyes (fluorochromes) as the reporter molecule.
Fluorescent dyes or fluorochromes are generally known and used for fluorescence labeling and detection of various biological and non-biological materials by procedures such as fluorescence microscopy, fluorescence immunoassay, and flow cytometry. A typical method for labeling such materials with fluorescent dyes is to create a fluorescent complex by means of bonding between suitable groups on the dye molecule and compatible groups on the material to be labeled. In this way, materials such as cells, tissues, amino acids, proteins, antibodies, drugs, hormones, nucleotides, nucleic acids, lipids and polysaccharides and the like may be chemically labeled and detected or quantified, or may be used as fluorescent probes which can bind specifically to target materials and be detected by fluorescence detection methods. Brightly fluorescent dyes permit detection or localization of the attached materials with great sensitivity.
Optical imaging with fluorescent dyes has emerged as a powerful imaging modality with significant advantages over other modalities both in vitro and in vivo. Dyes that fluoresce in the far red to near-infrared (NIR) region (630-900 nm) are essential for in vivo imaging due to the superior penetration of light through tissue at these wavelengths relative to longer and shorter wavelength light, which is absorbed by water and hemoglobin. NIR dyes also absorb and emit far outside of the typical range of tissue autofluorescence, making them extremely well suited for in vitro imaging of tissues and cells.
For many years, indocyanine dyes have been the dominant class of dyes used for NIR fluorescent imaging in vivo, with indocyanine green (molecular weight 775 Da) being one of the best known NIR dyes approved for diagnostic use in humans. In addition, numerous derivatized versions of indocyanines bearing various linking functionality such as carboxylic acids have been developed for use in bioconjugation and imaging applications. However, the current molecular constructs that are fluorescent in the NIR region, including the indocyanine family, tend to be large in size (>750 Da) and have poor solubility in water necessitating the incorporation of solubilizing groups such as multiple sulfonate groups. The resulting dyes then show very low cell membrane permeability, limiting their use for the targeting of intracellular structures.
There is an increasing need to develop novel, far red to NIR fluorescent fluorophores that are smaller and highly permeable to cell membranes so as to expand the reach of NIR imaging to intracellular targets, both in vitro and in vivo. The ideal fluorophores for such purposes would be small in size (<750 Da), have good water solubility, have absorbance and emission profiles in the far red to NIR range with high extinction coefficients and quantum yields, be highly permeable to the membranes of living cells and have tunable optical properties through variation of key substituents.
Notwithstanding, there is an ongoing need for new dyes that can be used in various medical, diagnostic and biological applications. There is a need for dyes that work well in in vitro, ex vivo and in vivo applications.